Project Summary Alzheimer's disease (AD) is a fatal neurodegenerative disorder that affects 10-30% of the population over 65 years old. The pathology of the disease is characterized by the failure to clear extracellular amyloid-? (A?) pep- tides and intracellular neurofibrillary tangles from the brain. In the continued absence of an AD therapy, the disease is persistent, disabling, costly and ultimately fatal. Microglia, the resident phagocytes of the central nervous system, play a major role in the clearance of A? aggregates. The activation state and phagocytosis capability of microglia are modulated by the CD33 cell-surface receptor. Specifically, the AD risk allele rs3865444 is associated with higher levels of CD33 that suppress microglial phagocytosis. In contrast, CD33 inactivation improves phagocytosis and mitigates A? pathology. CD33 inhibition may thus represent a novel therapy for AD and, to aid in this endeavor, the current proposals aims to understand the structural basis of CD33 signaling. To ascertain the mechanism of signal transduction across the cell membrane, in Aim 1 the TM domain border, the aggregation state of the TM domain, and the structure and dynamics of the TM and cyto- solic domains will be determined by solution NMR spectroscopy. To connect ligand binding and associated bio- logical effects with structural events that provide first insight into the mechanism of TM signaling, in Aim 2 the CD33-binding sites and affinities of two cytosolic ligands will be characterized by NMR and ITC. The binding sites further identify the structural and functional roles of the cytosolic receptor tail and provide insight into as- sembly of cytosolic signaling complexes. CD33 belongs to the family of sialic acid-binding immunoglobulin-like receptors (Siglecs) that regulate the function of cells in the innate and adaptive immune systems through the recognition of their glycan ligands. The proposed research provide basic insight into the little understood struc- tural basis of Siglec signaling, identifies targets sites and sequences for the pharmaceutical intervention in CD33 signaling and, thus, offers a direct avenue to therapeutic AD intervention.